Study On Regulation Of Targeting Ubiquitinated Protein Degradation Via Nano-optogenetic System

Targeting ubiquitinated protein degradation technology is a subversive biotechnology developed in recent years,which induces cell's inherent ubiquitinationproteasome system specifically knocks down the target protein directly at the posttranslation level.Compared with the traditional interfering protein function technology,the targeted ubiquitinated protein degradation technology could recognize the target protein specificly,knock down the target protein at the post-translation level directly,and not produce intermediate negative effects,which has shown broad application prospects in the field of biomedical research.However,the existing targeted ubiquitinated protein degradation technology is unable to accurately control the activity of protein or small molecule degrading agents,and has the defect that it is difficult to regulate the degradation process of the target protein,which prevents the technology from being converted into clinical applications.Therefore,on the basis of the existing technology,explore a safer,more precise,controllable new protein degradation technology has become the main direction of current research.Optogenetic is a technology that combines optical technology and genetic engineering technology.It has remarkable spatial and temporal precise control characteristics and is expected to meet the needs of regulating protein degradation.Based on this feature,this paper designed and constructed a nano-optogenetic system for regulating ubiquitin-mediated protein degradation.The core principle of the function of this system is: the use of optogenetics technology to synthesize lightsensitive bifunctional fusion protein Light ON/Nb-Fc,which regulates its intracellular expression by providing different light conditions,combined with TRIM21,so that the expressed fusion protein could specifically recognize the target protein and recruit the ubiquitination-proteasome system to achieve the degradation of target protein.This article mainly discusses the protein degradation effect of the system through the following content:1.In Chapter 2,in order to verify the feasibility of the system and optimize its related functions,we took green fluorescent protein(GFP)as the target protein and introduced the light-sensitive protein Light ON/vhh GFP4-Fc in combination with E3-TRIM21 has successfully constructed a light-regulated protein degradation system.The experimental results show that the system could degrade the target protein in different cell lines(Hela cells and 293 T cells)in vitro through the construction of blue LED related equipment,and could further control the ubiquitin-mediated protein degradation by changing the light time or light frequency.With the help of rare earth up-conversion nanomaterials as gene carriers and in vivo light sources,the degradation of target proteins could be regulated in vivo(in tumor models).2.In Chapter 3,in order to evaluate the practical application of the system,we took the non-structural protein 9(NSP9)that affects the reproduction of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)as the target protein,by introducing the light-sensitive protein Light ON/vhh Nb6-Fc used this nanophotogenetic system to regulate the degradation of NSP9.The experimental results show that the system could successfully degrade NSP9 protein and inhibit PRRSV reproduction under light conditions.By changing different light conditions,the degree of degradation of NSP9 could be adjusted to further regulate the reproduction status of PRRSV.In summary,we have successfully constructed a nano-photogenetic system for regulation ubiquitin-mediated protein degradation,verified the feasibility of the system through experiments,optimized its function,and further discussed the system's control the degradation of NSP9 has an impact on PRRSV reproduction.Our research makes up for the shortcomings of targeted protein degradation technology in regulation and meets the needs of targeted regulation of target protein degradation.We hope that this system could provide new ideas for the development of targeted protein degradation technology and help protein degradation technology to achieve clinical application and transformation.